The overall objective of the proposed studies is to understand how cleavage planes are specified in determinative cell divisions (divisions that produce daughter cells of differing size and developmental potential). Since the position of the mitotic apparatus dictates the cleavage plane, it is important to define the mechanisms governing spindle orientation. Previous studies suggest that spindle alignment is likely to involve interactions of the astral microtubules with the cell cortex. The focus of this study will be to characterize the dynamic interactions of microtubules with the cell cortex using in vivo fluorescence microscopy and multi-focal plane time-lapse recordings of developing Caenorhabditis elegans embryos. We will use these techniques to further examine, from the existing collection of maternal-effect embryonic lethal mutants, candidates defective in asymmetric cell division in order to identify genes required for cleavage plane specification. Finally, we will use in vivo imaging to examine the importance of local microtubule dynamics in spindle alignment and electron microscopy to obtain detailed images of the cortical microtubule attachment sites. Results from this study will help to define the basic mechanisms and molecular components that regulate cleavage plane specification in determinative cell divisions.